Abstract
ABSTRACT
Organoids are self‐organized, in vitro 3D structures derived from progenitor or pluripotent stem cells. During culture, these tissues secrete small extracellular vesicles (sEVs)—functional bioactive molecules—into their microenvironment. Here, we differentiated iPSC‐derived cerebral organoids (COs) under three culture conditions (static, orbital shaker, and a novel dynamic Organoid‐on‐a‐Chip platform) over 30 days of maturation to determine how culture dynamics shape organoid development and EV profiles. Transcriptomic analysis revealed strong, culture condition‐specific modulation of gene expression, while EV proteomics showed similar profiles across conditions at early maturation stages but marked divergence at later stages, reflecting differences in cellular maintenance, stress response, and tissue‐specific maturation pathways. Additionally, sEVs from all conditions reduced U87 glioblastoma cell viability and migration, with the strongest effect observed for sEVs derived from the orbital shaker condition. These findings demonstrate that organoid culture microenvironment substantially influences both developmental trajectory and the bioactivity of secreted EVs.